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21NIR SKULL OPTICAL CLEARING WINDOW FOR IN VIVO CORTICAL VASCULATURE IMAGING AND TARGETED MANIPULATION
DONG-YU LI1,2,3, ZHENG ZHENG4, TING-TING YU1,3, BEN ZHONG TANG4, PENG FEI5, JUN QIAN2 AND DAN
ZHU1,3
1Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science
and Technology, China
2State Key Laboratory of Modern Optical Instrumentation, Centre for Optical and Electromagnetic Research, College of Optical
Science and Engineering, Zhejiang University, China 3MoE Key Laboratory for Biomedical Photonics, Huazhong University of Science and Technology, China 4Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction Division of Life Science, State Key Laboratory of Molecular Neuroscience, Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, The Hong Kong University of Science and Technology, China 5School of Optical and Electronic Information-Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and
Technology, China
dawnzh@mail.hust.edu.cn
Abstract
In vivo observation of brain in its natural environment is of vital significance to better understand the function of vasculature and neural networks, as well as various diseases related to the dysfunction of brain [1-3].Modern optical imaging technology combined with a variety of fluorescent labelling technology can obtain the structure and function information of biological tissue with high spatial and temporal resolution, providing an important means for brain science [4-6].However, the high scattering characteristic of skull limits the penetration depth of light[7, 8]. Since the scattering of tissue decreases with the increase of wavelength[9], the imaging in near infrared band, especially in the second region of near infrared, shows great advantages for improving the ability of optical imaging in deep tissue imaging[10-16].Compared to two-photon fluorescence or second harmonic generation, three-photon fluorescence or third harmonic generation (THG) based on longer wavelength excitation, can obtain deeper information with high resolution.
The recent development of tissue optical clearing technology reduces the effect of scattering from another perspective, providing a new idea for deep tissue imaging.With the novel skull optical clearing window, optical imaging techniques, laser speckle contrast imaging, hyperspectral imaging and two-photon fluorescence microscopy have been applied to observe cortical neuron, microglia, vascular structures and functions [17-21].Since NIR-II excitation based nonlinear optical microscopy and skull optical clearing are useful means, respectively, to realize in vivo cortical imaging without craniotomy, does the combination of the both have an enhancement effect? After all, the previous optical clearing windows only demonstrated the efficacy of optical imaging in the wavelength range of visible to NIR-I [17, 18].
Figure 1. (a) Transmission spectra of D2O (red line) and H2O (blue line). (b) Transmission spectra of S1 of VNSOCA (red line) and USOCA (blue line). (c) Transmission spectra of S2 of VNSOCA (red line) and USOCA (blue line). (d) THG images of glass capillaries filled with DCCN nanocrystal dispersion with the 25x objective immersed in S1 and S2 of VNSOCA and USOCA respectively. The dashed lines represent the edges of the capillaries. (e) The THG intensities of DCCN nanocrystal dispersion filled in capillaries with the objective immersed in S1 and S2 of VNSOCA and USOCA respectively.
In this work, we systematically studied the combination of NIR-II excited THG microscopyand in vivotissue optical clearing technique, and further developed Vis-NIR-II compatible optical clearing skull window. Compared with the
10
previous urea-based skull optical clearing agent (USOCA), the newly developedvis-NIR-II optical clearing agent (VNSOCA) not only had the same transmittance in the shorter wavelength range, but also hadgreatly enhanced transmittance in the near infrared region (Fig. 1).
The optical clearing window could remarkably increase signal intensity as well as the imaging depth of cortical THG vascular imaging (Fig. 2 and Fig. 3). Finally, the imaging depth of 650^m was obtained (Fig. 4), which was even close to it without skull [22].
Position (mm) Position (mm) Bar: 200
Figure 2. (a, b) Typical white-field images of cortical blood vessels before and after VNSOCA treated. (c) Intensity profiles along the red dashed lines across the vasculature in (a): black, and (b): red. The arrows indicate vessels those couldn't be observed before VSOCA treated. (d,e) Typical large-field THG images collected with a 5x scan lens before and after skull clearing. f) Intensity profiles along the red dashed lines across the vasculature in (d): black, and (e): red. The arrows indicate vessels those couldn't be observed before VSOCA treated. (g-i) THG microscopic images of various areas in (e), using a 25x objective. The frame color was used to represent the congruent relationship.
DO covered VNSOCA liculcrj
Figure 3. (a-d) THG scanning microscopy at different depth using the 25x objective without skull clearing. (e-h) THG scanning microscopy at different depth using the 25x objective with skull clearing. (i-l) Intensity profiles along the white dashed lines across the vasculature in (a-h), respectively. (m) THG imaging of cortical vasculature at particular imaging depths. (n-p) 3D reconstruction of vasculature in certain volumes.
In addition of imaging, the effectiveness of optical manipulation is also significant.The results showed that precise NIR-II light manipulation could be performed through the established skull optical clearing window. Using the 1550-nm fs laser, both large vessel or small capillary could be targeted injured (Fig. 4).
Partial Scanning
Partial Scanning
Figure 4. Dynamically observing cerebral hemorrhage using THG scanning imaging. The cerebral hemorrhage was made by partically scanning the region showed by the red circles for (a) 10 s and (b) 15 s.
The novel Vis-NIR-II optical clearing skull window is well adapted to the whole band from visible light to NIR-II region,
which greatly expands the wavelength selection range of deep cortex optical imaging and can effectively combine
various NIR-II imaging technologies developed in recent years.The first established scalp-cranial window model further
simplifies the cranial window model and provides a new approach for imaging the transcranial cortex in vivo.
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